Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.

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Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.

Ewelina Celińska, Rodrigo Ledesma Amaro, Macarena Larroudé, Tristan Rossignol, Cyrille Pauthenier, Jean-Marc Nicaud

To cite this version:

Ewelina Celińska, Rodrigo Ledesma Amaro, Macarena Larroudé, Tristan Rossignol, Cyrille Pauthe- nier, et al.. Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.. Microbial Biotechnology, Wiley, 2017, 10 (2), pp.450-455. �10.1111/1751-7915.12605�.

�hal-01602609�

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Brief report

Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica

Ewelina Celi nska,

^1,^†

Rodrigo Ledesma-Amaro,

^2,

*

^,^†

Macarena Larroude,

²

Tristan Rossignol,

²

Cyrille Pauthenier

³

and Jean-Marc Nicaud

^2,

*

1

Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-627 Poznan, Poland.

2

Micalis Institute, INRA, AgroParisTech, Universit e Paris- Saclay, 78350 Jouy-en-Josas, France.

3

Institute of System and Synthetic Biology, Universite d’Evry vald’Essonnes, Bt. Geneavenir 6 Genopole Campus 1, 5 rue Henry Desbrueres 91000, Evry, France.

Summary

In this study, we have adopted Golden Gate modu- lar cloning strategy to develop a robust and versa- tile DNA assembly platform for the nonconventional yeast

Yarrowia lipolytica. To this end, a broad set of

destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen ele- ments. Previously validated DNA modules (regula- tory elements and selection markers) were adopted as the Golden Gate bricks. The system’s operability was demonstrated based on synthetic pathway of carotenoid production. This technology greatly enriches a molecular biology toolbox dedicated to

this industrially relevant microorganism enabling fast combinatorial cloning of complex synthetic pathways.

Introduction

Synthetic biology, and in particular combinatorial cloning, derives from engineering concepts of standardization, modularity of building blocks and simplification of assem- bly lines. Traditional protocols employing restriction digestion and one-by-one element cloning are both time- and cost-inefficient (Celi nska and Grajek, 2013; Mat- thaus et al., 2014). Due to the recent development of DNA assembly techniques for metabolic pathway engi- neering, a great worldwide effort is now being pursued towards establishing such cloning platforms for an indi- vidual organism of interest. In the last years, several DNA modular assembly platforms have been developed and new standards have been defined (Sands and Brent, 2001). Golden Gate (GG) modular cloning system, relying on type IIs restriction enzymes, appears as one of the most robust techniques within this

ﬁeld (Engler

et al., 2008; Gao et al., 2013). The core of GG strategy lies in establishing a library of standardized and inter- changeable DNA parts, which can be subsequently assembled in a single-step, one-pot reaction. Examples of such GG platforms have been recently reported for Escherichia coli, yeast or plant species (Engler et al., 2014; Terfr€ uchte et al., 2014; Agmon et al., 2015; Kakui et al., 2015; Lee et al., 2015; Mitchell et al., 2015; Iver- son et al., 2016; Moore et al., 2016). Nevertheless, many of valuable biotech workhorses are still queuing the line for a customized GG platform, amongst these the non-conventional yeast Y. lipolytica, which is a well- established biotechnological chassis for the production of numerous valuable bioproducts (Nicaud, 2012;

Ledesma-Amaro et al., 2015; Madzak, 2015; Ledesma- Amaro and Nicaud, 2016). Establishing a GG combinato- rial cloning platform for Y. lipolytica appears as an urgent need, enabling more time- and cost-efﬁcient assembly of complex DNA constructions and standard- ization of DNA modules, which can be easily exchanged between different laboratories.

Received 4 November, 2016; revised 30 December, 2016; accepted 4 January, 2017. *For correspondence. E-mail rodrigo.ledesma- amaro@inra.fr; Tel. (33) 01 74 07 16 92;

Fax 0134652088; (RL-A) and E-mail jean-marc.nicaud@ inra.fr;

Tel. (33) 01 74 07 18 20; Fax 0134652088 (J-MN).

†These authors contributed equally to this work.

BIMLip, Biologie integrative du Metabolisme Lipidique team (RLA, ML, TR, JMN).

Microbial Biotechnology(2017)10(2), 450–455 doi:10.1111/1751-7915.12605

Funding Information Agence Nationale de la Recherche (ANR- 11-BTBR-0003), Agreenskills, (FP7-267196), European Molecular Biology Organization (EMBO_ASTF_117-2016).

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Here, we present a robust modular GG system for expression of one, two or three customizable transcrip- tion units (TU) in a versatile cassette for Y. lipolytica.

Results and discussion

Deﬁning standards and modular parts of the GG system for Y. lipolytica

The ultimate goal of this study was the development of extensive and versatile system of building blocks (Golden Gate Fragments; GGFs) easily

ﬁtted into the

general scaffold of GGA (Golden Gate Assembly), enabling fast construction of complex expression vec- tors dedicated to metabolic engineering of Y. lipolytica.

To complete this task, a set of thirteen 4 nt overhangs was developed together with the corresponding

destination vector system (Fig. 1, Supporting Informa- tion) to cover three TUs (all composed of promoter

–

P; ORF

=

gene

–

G; terminator

–

T; all accompanied with a sufﬁx indicating position of a TU); selection marker gene

–

M; and integration targeting sites

–

InsUP and InsDOWN.

Incompatibility of the 4 nt sequence was a prerequisite to avoid the risk of unintended matching of the building blocks in uncontrolled position and/or orientation. Such a system of the overhangs was developed and organized in a general scaffold (Fig. 1). Due to extensive character of the envisioned expression cassettes, each of the overhangs was assigned a letter, to enable unambigu- ous referencing to the respective position in the GGA.

Moreover, the overhangs

ﬂanking individual genes (in

position G1, G2 or G3; see Fig. 1) were designed to

A:InsUP:B B:M:C C:P1:D D:G1:E E:T1:F F:P2:G G:G2:H H:T2:I I:P3:J J:G3:K K:T3:L L:InsDOWN:M

zeta _SﬁI zeta_NotI pLip2 pUra3

URA3 LEU2 LYS5 Hyg

pTEF pPGM pGAPdh 2UASpTEF 4UASpTEF 8UASpTEF

GGS1 pTEF

pPGM pGAPdh 2UASpTEF 4UASpTEF 8UASpTEF pTEF

pPGM pGAPdh 2UASpTEF 4UASpTEF 8UASpTEF tTEF

tXPR2 tLip2 tGuo tsynth27 tsynth8

tTEF tXPR2 tLip2 tGuo tsynth27 tsynth8

zeta _SﬁI zeta_NotI tLip2 tUra3

carB carRP

KanR KanR KanR KanR KanR KanR KanR KanR KanR KanR KanR KanR

NotI or SfiI

ori AmpR/CmR/KanR

InsUP M P1 G1 T1-3 InsDOWN

NotI or SfiI A: GCCT

B: AGGT

D: AATG E: TCTA L: GAGT

M: TGCG

NotI or SfiI

ori

InsUP M P1 G1 T1 P2 G2 T2-3 InsDOWN

B: AGGT

D: AATG E: TCTA F: GCTT

G: ACAA H: GGAT

L: GAGT

M: TGCG

NotI or SfiI

ori

InsUP M P1 G1 T1 P2 G2 T2 P3 G3 T3 InsDOWN

B: AGGT C: ACGG

D: AATG E: TCTA F: GCTT

G: ACAA

H: GGAT I: GTCA

J: CCAC K: GTAT

L: GAGT

M: TGCG

AmpR/CmR/KanR

1-TU-bearing GGA 2-TU-bearing GGA

3-TU-bearing GGA

C: ACGG C: ACGG

(A)

(B)

AmpR/CmR/KanR

Fig. 1.Golden Gate Assembly platform dedicated toYarrowia lipolyticain a one-, two- and three-transcription unit (TU)-bearing format.

A. Schematic representation of complete GG destination vectors in a one-, two- and three-TU-bearing format. Each of GGA covers at least one TU (composed of promoter–P; gene as ORF–G; and terminator–T) and selection marker (M), which areflanked with integration targeting sequences (InsUP and InsDOWN), constructed on a destination vector backbone, either pSB1A/C/K3-RFP/amilCP/amilGFP for ampicillin (A), chloramphenicol (C) or kanamycin (K) resistance marker respectively; pSB vector series offers exploitation of one of the following reporter genes inE. coli: RFP–red chromophore; amilCP–blue chromophore; amilGFP–greenfluorophore. Building blocks areflanked with predesigned 4 nt overhang, each assigned a letter from A–M. Sequences of the overhangs are provided next to the respective position. Abbrevia- tions: InsUP/DOWN–sequence targeting insertion in the 5’/3’region; M–selection marker; P1/2/3–promoter governing expression of the gene of interest in transcription unit 1, 2 and 3 respectively; G1/2/3–gene of interest in transcription unit 1, 2 and 3 respectively; T1/2/3–terminator located in the transcription unit 1, 2 and 3 respectively. NotI/SfiI: NotI/SfiI recognition sites for release of the expression cassettes prior to Y. lipolyticatransformation. B. Donor vectors constructed on a backbone of pCR II-Blunt TOPO vector (Invitrogen/Life Technologies) bearing kanamycin resistance gene. Each building block isflanked with respective BsaI recognition sites and predesigned 4 nt overhangs. Exemplary building blocks (Golden Gate Fragments; GGFs) constructed in this study are provided as a list underneath the donor vectors’schemes. For reference to nucleotide sequences, see Supporting Information.

ª 2017 The Authors.Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology,Microbial Biotechnology,10, 450–455

Golden Gate platform for Yarrowia lipolytica 451

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minimize the scar between a coding sequence and the regulatory elements. Thus, the overhang prior to the cod- ing sequence was designed to maximally

ﬁt into the con-

sensus sequence preceding signiﬁcant fraction of ORFs in Y. lipolytica genome

–

CACA

–

or include the start codon (Gasmi et al., 2011).

The GG modular system was designed to include two

ﬂanking insertion sites, a selection marker and one, two

or three TUs in each assembly. The number of TUs can be easily manipulated by ampliﬁcation of

‘hybrid’

termi- nators, provided that the same sequence was used in the merged positions (T1, T2, T3; compare Fig. 1).

These

‘hybrid’

terminators should be ampliﬁed with for- ward primer with

‘E’

overhang and reverse primer with

‘L’

overhang to render T1-3

‘hybrid’

terminator, skipping the second and third transcription units, or with

‘H’

over- hang bearing forward primer and

‘L’

overhang bearing reverse primer, to gain T2-3

‘hybrid’

terminator, skipping the third transcription unit.

The destination vector backbones were equipped with a chromophore (RFP or amilCP) or

ﬂuorophore

(amilGFP) gene

ﬂanked with BsaI sites with pre-

designed overhangs to deﬁne a colour-based negative cloning selection marker. The BsaI recognition sites were positioned outwardly with respect to the back- bone of the destination vector, so that after digestion with BsaI, both the reporter gene and the BsaI recog- nition sites were released, leaving the vector’s back- bone

ﬂanked with protruding 4 nt overhangs. A set of

different antibiotic resistance genes was incorporated into the system of the destination vectors, to enable greater

ﬂexibility of experimental set-up. In the proof-

of-concept experiments (with three TUs bearing cas- sette; described below), we have used a pSB1K3-RFP variant from the iGEM collection (http://parts.igem.org/

Collections).

It is commonly accepted that only integrative expres- sion cassettes constitute truly potent tools in Y. lipolytica genetic engineering, as the so far developed episomal plasmids tend to be unstable and low-copy (Liu et al., 2014). This fact ruled the construction of both the desti- nation plasmids as well as the scaffold of GGA,

ﬂanked

with GGFs targeting integration of the expression cas- sette into the host genome (InsUP and InsDOWN).

Thus, the destination vector’s backbone containing solely bacterial elements can be discarded prior to transforma- tion of Y. lipolytica cells. To enable the release of the expression cassette composed of transcription units, selection marker and targeting sequences, from the bac- terial backbone, rare-cutting restriction endonuclease recognition sites NotI or SﬁI, depending on the targeted GGA structure, were added at the borders of the expres- sion cassette in the InsUP and InsDOWN fragments (Fig. 1).

Generation of GG library dedicated to Y. lipolytica All the sequences to be used as building blocks of the envisioned GGA were extracted from Y. lipolytica W29 genome sequence or one of the previously constructed vectors from our own collection. All the sequences were analysed in silico to

ﬁnd internal BsaI site, to be eventu-

ally eliminated using assembly PCR technique (Sam- brook and Russell, 2001). Subsequently, an extensive set of primers (referred to as GGP, for Golden Gate Pri- mers) equipped with the predesigned system of the 4 nt overhangs and externally located BsaI recognition sites was designed (Supporting Information; Pauthenier and Faulon, 2014). The set of GGF presented in this work, the interchangeable parts for promoter, terminator, selec- tion marker and integration site (Fig. 1B), constitutes the response to our current needs, but can be easily expanded on the other building blocks of interest by sim- ply adding the required prefixes and suffixes within the primers at the stage of the element amplification. As pre- sented in Fig. 1, the major focus of this study was to develop a system of easily tractable phenotype of posi- tive transformants. Therefore, for the genes of interest we have chosen the complete pathway for carotenoid production, for convenient traceability of positive clones.

With the same concept in mind, LIP2 and URA3 loci were selected as the targeting platforms, and thus, cor- rect integration could be veriﬁed on tributyrin- or sucrose-containing plate respectively. A LIP2 deletion results in a reduced halo of triglyceride hydrolysis (Pign ede et al., 2000), while replacement of ura3-320 allele bearing S. cerevisiae SUC2 gene gives transfor- mants unable to grow on sucrose (Nicaud et al., 1989;

Barth and Gaillardin, 1996). Zeta sequences were cho- sen to reach random integrations in Po1d strain (JMY195) or at a zeta docking platform in Po1d deriva- tive JMY1212, bearing a zeta sequence at the leu2-270 locus (Bordes et al., 2007).

All the amplicons bearing respective GGFs were cloned in the donor vectors (pCR Blunt II TOPO vectors;

Thermo Fisher Scientiﬁc, Villebon sur Yvette, France), veriﬁed by BsaI digestions and sequencing and depos- ited in our collection. The structure of the targeted GGA cassette ruled the composition of the GG reaction mix- ture. The Golden Gate reaction conditions were designed based on the previously published protocols (Engler et al., 2008; Pauthenier et al., 2012; Agmon et al., 2015). The reaction mixture contained precalcu- lated equimolar amount of each GGF and the destination vector (50 pmoles of ends), 2

ll of T4 DNA ligase buffer

(NEB), 5 U of BsaI, 200 U of T4 and ddH2O up to 20

ll.

The following thermal proﬁle was applied: [37°C for

5 min, 16°C for 2 min]

9

60, 55°C for 5 min, 80°C for

5 min, 15°C

∞. Subsequently, the reaction mixture was

452 E. Celi nska et al.

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used for E. coli DH5a transformation (Sambrook and Russell, 2001). White colonies were screened for identiﬁ- cation of complete GGA through plasmid isolation, restriction digestion and multiplex PCR. Complete GGA was subsequently linearized and used for transformation of either Y. lipolytica Po1d ura- leu- (JMY195 MatA leu2- 270 ura3-302 xpr2-322) or Po1d ura- leu+ bearing a zeta docking platform (JMY1212; MatA ura3-302 xpr2-322, LEU2, zeta; Bordes et al., 2007) via the lithium acetate transformation protocol (Chen et al., 1997). Derivative strain Po1d leu+ ura+ (JMY2900; MatA ura3-302 xpr2- 322) was used as prototroph control. Clones bearing complete GGA integrated with the host genome exhib- ited red–orange phenotype due to carotenoid production.

All the GGF building blocks included in the GGA have been proven functional in previous experiments (see Table S1).

Proof of concept based on carotenoid-pathway- containing Golden Gate Assembly

To validate robustness and efﬁciency of the here devel- oped GG system, we have constructed a complex assembly structure comprising twelve GGFs and the destination vector, covering three TUs, accompanied by selection marker and integration targeting sequences (Fig. 2A).

The three genes within this assembly were native GGS1 (geranylgeranyl diphosphate synthase;

YALI0D17050g) in G1 position and heterologous carB (phytoene dehydrogenase; AJ238028.1) and carRP (phytoene synthase; AJ250827.1) coding sequences from Mucor circinelloides in G2 and G3 positions respec- tively (sequences were kindly provided by DSM

Company). Such a set of genes was previously proved to be efﬁciently expressed in Y. lipolytica cells, con- structed using DNA assembler strategy (Gao et al., 2014) or classical cloning techniques (Matthaus et al., 2014). All the genes were assembled with corresponding regulatory elements, zeta insertion sites and URA3 selection marker. The correct complete GGA was

ﬁrst

preselected in E. coli clones through multiplex PCR and restriction digestion and then linearized and transformed into competent Y. lipolytica JMY195 cells. On average, more than 4

9

10

²

colonies could be obtained from a single transformation reaction. About 10% of the trans- formants were white (exemplary results for one transfor- mation run: 50/517, 91/1052, 41/396). The remaining clones exhibited different intensity of the orange colour development (Fig. 2B). The observed variability in the carotenoid-producer phenotype could result from varying number of either complete or partial GGA copies inte- grated within the host genome as observed by Gao et al.

(2014), although this would be unlikely with the amount of DNA we used (Bordes et al., 2007). More likely, the random distribution of the cassette within the genomic DNA due to the zeta targeting sequences, as investi- gated in Pignede et al. (2000), may be responsible for the colour variability. This hypothesis is supported by the results obtained when the same GGA was used to trans- form strain JMY1212, bearing a zeta docking platform at the leu2-270 locus enabling homologous recombination.

Although the number of white colonies after transforma- tion of this strain was higher (33%; 33/98), the orange colonies did not present the colour variability that was found amongst JMY195-based transformants. In a previ- ous study with DNA assembler strategy, when the same carotenoid-pathway genes were used, the efﬁciency of

A:zeta:B B:ura3:C C:pTEF:D D:GGS1:E E:tLip2:F F:pTEF:G G:carB:H H:tLip2:I I:pTEF:J J:carRP:K K:tLip2:L L:zeta:M

KanR KanR KanR KanR KanR KanR KanR KanR KanR KanR KanR KanR

SfiI

ori AnbiocR

(A)zeta ura3 pTEF GGS1 tLip2 pTEF carB tLip2 pTEF carRP tLip2 zeta

SfiI

(B)

Fig. 2.Golden Gate Assembly bearing‘carotenoid synthesis’pathway dedicated to integration withYarrowia lipolyticagenome.

A. Scaffold of GGA comprising three transcription units and selection marker,ﬂanked with integration targeting sequences, constructed on a destination vector backbone. Each gene wasﬂanked with 396 nt of TEF promoter and 122 nt of Lip2 terminator sequences, both native to Y. lipolytica.URA3(1289 nt) gene was used as selection marker in this assembly. Random integrations in Po1d strain (JMY195) were driven through zeta sequences (305 nt and 395 nt for UP and DOWN respectively). Representation of donor vectors bearing respective GGFs is provided below. B.Y. lipolyticaJMY195 transformants bearing‘carotenoid synthesis’GGA. Variability in colour development is discussed in the Results and Discussion section.

Golden Gate platform for Yarrowia lipolytica 453

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reaching desired phenotype was 20% (Gao et al., 2014).

In the current study, the efﬁciency was largely improved, as 67% to 90% of the obtained Y. lipolytica clones exhibited desired red–orange phenotype, for JMY1212 and JMY195 strains respectively. Improved efﬁciency was accompanied by time- and workload savings, as well as versatility and standardization, being indigenous for modular DNA cloning techniques, altogether making this approach a method of choice for synthetic biology endeavours.

Here, we present a Golden Gate standard speciﬁ- cally designed to modify Y. lipolytica in a versatile manner. Robustness and reliability of the system were validated for thirteen-element-bearing GGA, with high efﬁciency of the desired phenotype recovery in the recombinants. This technology greatly enriches the molecular biology toolbox dedicated to this industrially relevant microorganism, permitting faster and accurate multiple target engineering, a limitless expansion of the building blocks library from endogenous or heterolo- gous origins, or the use of interchangeable modules in combinatorial approaches followed by screening desired phenotypes.

Acknowledgements

This study was

ﬁnancially supported by a programme

with the reference ANR-11-BTBR-0003, Agence Natio- nale de la Recherche, under the

‘Investissements d’ave-

nir’. RL would like to thank AgreenSkills Fellowship (FP7-267196), funded by European Union Marie-Curie FP7 COFUND People Programme and the Synthetic Genome Summer Course 2016. EC (EMBO_ASTF_117- 2016) would like to thank European Molecular Biology Organization for supporting her fellowship at the BIMLip team.

Authors’ contribution

EC and RLA contributed equally to this work. EC, RLA, ML and TR contributed experimental data. CP partici- pated in the selection of the 4 nt overhangs for the GGA scaffold and provided destination vectors. EC, RLA and JMN wrote the manuscript. The work was performed in the laboratory of TR and JMN.

Conﬂict of interest

The authors do not have any conﬂict of interest.

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Supporting information

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Table S1. A list of plasmids and oligonucleotides used in this study.